Pattern Recognition Analysis for Classification of Hypertensive Model Rats and Diurnal Variation Using 1H-NMR Spectroscopy of Urine

Abstract

IntroductionNMR-based Metabolomic analysis has mainly been used for toxicology or drug discovery.1,2 Significant metabolic responses have been detected on various bio samples with the dose, injection of toxins or high-salt diet. Recently, various life-style related diseases, such as hypertension, diabetes, and hyperlipidemia, have become serious topics of concern. To acquire potential information on disease status, including presymptomatic data, the detection of any profile difference from the normal in natural conditions is essential. To understand the progress of disease metabolism, we performed an experiment with 20 model rats: 10 rats that were spontaneously hypertensive (SHR/Izm) and 10 rats that were normal (Wistar Kyoto, WKY). The two strains' urine samples were collected under conditions of minimized intentional stress, such as on impact, dose, special diet and fasting. 3,4 The 1D 1 H-NMR spectra for the urine samples were measured. All of the spectra were analyzed by a pattern recognition method and classified by their pattern differences. It is notable that there is no need to assign each peak or to identify the compounds in the spectrum, which can be recognized as a profile histogram. 5 To investigate the reflection of the metabolic balance in excretions, the pattern recognition method is a faster and more straightforward approach than conventional NMR structure analysis. Experimental Animal handling and sample collectionTen rats each of 9-week-old males of WKY and SHR/Izm strain were purchased from SHIMIZU Laboratory Supplies Co., Ltd. The rats were housed by Bio-Science Center of KAC Co., Ltd. (Kyoto, Japan) in metabolic cages for 1 week acclimatization. All of the studies were conducted using the same basic protocol in that a standard solid diet, CRF-1 (Oriental Yeast Co. Ltd.) was given to all animals and free access to food and water was permitted. A temperature of 23 ± 2˚C and a relative humidity of 50 ± 10% were maintained with a 12 h light/12 h dark cycle. Urine samples were collected from each 10-week-old of WKY strain (average b.p. 105/75 mmHg, wt 265 g) and SHR/Izm strain (average b.p. 171/140 mmHg, wt 255 g), during the daytime (14:00 -18:00) and nighttime (18:00 -9:00), respectively. The samples were sent to us maintained at 0 -4˚C, and then measured within 2 days. In our laboratory, after centrifuging, 200 µL of a phosphate buffer solution was added to 400 µL of rat urine, with which 5% D2O and 0.1% (w/v) NaN3 were mixed, to obtain pH 7.4. Each 600 µL of the solution was used for an NMR measurement. NMR spectroscopyThe 1 H NMR spectra were acquired using a 500 MHz JEOL ECA spectrometer at 25˚C. Each spectrum consisted of 16 K complex data points with a spectrum width of 5 kHz, where each spectrum was accumulated by 128 scans with an acquisition time of 1.75 s, and a recycle delay of 5 s per scan. The detection pulse flip angle was set to 45˚. A pre-saturation sequence was used to suppress the water signal. NMR data reduction procedures and pattern recognition analysisEach NMR spectrum wa...

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